Racemization at the Asp 58 residue in αA‐crystallin from the lens of high myopic cataract patients

Post‐translational modifications in lens proteins are key causal factors in cataract. As the most abundant post‐translational modification in the lens, racemization may be closely related to the pathogenesis of cataract. Racemization of αA‐crystallin, a crucial structural and heat shock protein in the human lens, could significantly influence its structure and function. In previous studies, elevated racemization from l ‐Asp 58 to d ‐isoAsp58 in αA‐crystallin has been found in age‐related cataract (ARC) lenses compared to normal aged human lenses. However, the role of racemization in high myopic cataract (HMC), which is characterized by an early onset of nuclear cataract, remains unknown. In the current study, apparently different from ARC, significantly increased racemization from l ‐Asp 58 to d ‐Asp 58 in αA‐crystallin was identified in HMC lenses. The average racemization rates for each Asp isoform were calculated in ARC and HMC group. In ARC patients, the conversion of l ‐Asp 58 to d ‐isoAsp 58, up to 31.89%, accounted for the main proportion in racemization, which was in accordance with the previous studies. However, in HMC lenses, the conversion of l ‐Asp 58 to d ‐Asp 58, as high as 35.44%, accounted for the largest proportion of racemization in αA‐crystallin. The different trend in the conversion of αA‐crystallin by racemization, especially the elevated level of d ‐Asp 58 in HMC lenses, might prompt early cataractogenesis and a possible explanation of distinct phenotypes of cataract in HMC.     

Improved high-performance liquid chromatographic analysis of terazosin in human plasma

A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method was developed for the determination of terazosin in human plasma. The method involves a one-step single solvent extraction procedure using dichloromethane with a 0.25 ml plasma sample. Recovery values were all greater than 90% over the concentration range 0.25–100 ng/ml. Terazosin was found to adsorb to glass or plastic tubes, but this could be circumvented by using disposable plastic tubes. Also, rinsing the injector port with methanol after each injection helped to prevent any carry-over effect. The internal standard, prazosin, did not exhibit this problem. The method has a quantification limit of 0.25 ng/ml. The within- and between-day coefficient of variation and accuracy values were all less than 7% over the concentration range 0.25–100 ng/ml and hence the method is suitable for use in pharmacokinetic studies of terazosin.     

Determination of the rate and yield of B-side quinone reduction in Rhodobacter capsulatus reaction centers

In the native purple bacterial reaction center (RC), light-driven charge separation utilizes only the A-side cofactors, with the symmetry related B-side inactive. The process is initiated by electron transfer from the excited primary donor (P*) to the A-side bacteriopheophytin (P* --> P+ H(A)-) in approximately 3 ps. This is followed by electron transfer to the A-side quinone (P+ H(A)- --> P+ Q(A)-) in approximately 200 ps, with an overall quantum yield of approximately 100%. Using nanosecond flash photolysis and RCs from the Rhodobacter capsulatus F(L181)Y/Y(M208)F/L(M212)H mutant (designated YFH), we have probed the decay pathways of the analogous B-side state P+ H(B)-. The rate of the P+ H(B)- --> ground-state charge-recombination process is found to be (3.0 +/- 0.8 ns)(-1), which is much faster than the analogous (10-20 ns)(-1) rate of P+ H(A)- --> ground state. The rate of P+ H(B)- --> P+ Q(B)- electron transfer is determined to be (3.9 +/- 0.9 ns)(-1), which is about a factor of 20 slower than the analogous A-side process P+ H(A)- --> P+ Q(A)-. The yield of P+ H(B)- --> P+ Q(B)- electron-transfer calculated from these rate constants is 44%. This value, when combined with the known 30% yield of P+ H(B)- from P in YFH RCs, gives an overall yield of 13% for B-side charge separation P* --> P+ H(B)- --> P+ Q(B)- in this mutant. We determine essentially the same value (15%) by comparing the P-bleaching amplitude at approximately 1 ms in YFH and wild-type RCs.     

Asymmetric cell division and Notch signaling specify dopaminergic neurons in Drosophila

In Drosophila, dopaminergic (DA) neurons can be found from mid embryonic stages of development till adulthood. Despite their functional involvement in learning and memory, not much is known about the developmental as well as molecular mechanisms involved in the events of DA neuronal specification, differentiation and maturation. In this report we demonstrate that most larval DA neurons are generated during embryonic development. Furthermore, we show that loss of function (l-o-f) mutations of genes of the apical complex proteins in the asymmetric cell division (ACD) machinery, such as inscuteable and bazooka result in supernumerary DA neurons, whereas l-o-f mutations of genes of the basal complex proteins such as numb result in loss or reduction of DA neurons. In addition, when Notch signaling is reduced or abolished, additional DA neurons are formed and conversely, when Notch signaling is activated, less DA neurons are generated. Our data demonstrate that both ACD and Notch signaling are crucial mechanisms for DA neuronal specification. We propose a model in which ACD results in differential Notch activation in direct siblings and in this context Notch acts as a repressor for DA neuronal specification in the sibling that receives active Notch signaling. Our study provides the first link of ACD and Notch signaling in the specification of a neurotransmitter phenotype in Drosophila. Given the high degree of conservation between Drosophila and vertebrate systems, this study could be of significance to mechanisms of DA neuronal differentiation not limited to flies.     

Regulation of Alr1 Mg transporter activity by intracellular magnesium

Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni(2+), Mn(2+), Zn(2+) and Co(2+). Using Ni(2+) uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms.     

Autophosphorylation of the Smk1 MAPK is spatially and temporally regulated by Ssp2 during meiotic development in yeast

Smk1 is a meiosis-specific MAPK that controls spore wall morphogenesis in Saccharomyces cerevisiae. Although Smk1 is activated by phosphorylation of the threonine (T) and tyrosine (Y) in its activation loop, it is not phosphorylated by a dual-specificity MAPK kinase. Instead, the T is phosphorylated by the cyclin-dependent kinase (CDK)–activating kinase, Cak1. The Y is autophosphorylated in an intramolecular reaction that requires a meiosis-specific protein named Ssp2. The meiosis-specific CDK-like kinase, Ime2, was previously shown to positively regulate Smk1. Here we show that Ime2 activity is required to induce the translation of SSP2 mRNA at anaphase II. Ssp2 protein is then localized to the prospore membrane, the structure where spore wall assembly takes place. Next the carboxy-terminal portion of Ssp2 forms a complex with Smk1 and stimulates the autophosphorylation of its activation-loop Y residue. These findings link Ime2 to Smk1 activation through Ssp2 and define a developmentally regulated mechanism for activating MAPK at specific locations in the cell.     

An ultrafiltration catheter for monitoring of venous lactate and glucose around myocardial ischemia

Early detection of myocardial ischemia is of major importance in critical-care medicine. Changes of lactate or glucose levels in the cardial venous efflux may be useful parameters. We succeeded in integrating an ultrafiltration membrane in a cardiac catheter for continuous sampling. The ultrafiltrate was analyzed outside the body, resulting in a lag-time of about 24 min. Biosensors in a flow-injection analysis system were used for minute by minute sample analyses. The coronary sinus of pigs was catheterized to monitor the effects of 5, 15 or 45 min ischemia by coronary artery obstruction or myocardial stress by dobutamine infusion. A total of 27 h was monitored. The intravascular response time was 1.33+/-0.61 min (10-90%). Linear regression in vivo of blood and ultrafiltrate samples was 0.977 for lactate and 0.994 for glucose. Lactate levels rose 0.38+/-0.10 mM above baseline within 5 min after ischemia. Reperfusion was clearly marked by a promptly peaking lactate release (maximum 9.27 mM). Myocardial stress by dobutamine increased glucose but not lactate levels. Once, a wall effect was noted at the catheter tip. In vivo semi-continuous myocardial monitoring of absolute lactate and glucose concentrations was thus achieved by an ultrafiltration catheter. Ischemia and reperfusion can be detected very early by a lactate level rise. Further, development of the ultrafiltration catheter will be focused on the diagnostic potential of lactate monitoring for patients.     

Association between public media and trends in new acute coronary syndrome presentations during the first COVID‑19 wave in the Netherlands

BackgroundWe aimed to evaluate the association between public media and trends in new presentations of acute coronary syndrome (ACS) during the first wave of the coronavirus disease 2019 (COVID‑19) in the Netherlands.MethodsNew ACS presentations per week in 73 hospitals during the first half of 2019 and 2020 were retrieved from the national organisation Dutch Hospital Data and incidence rates were calculated. Stratified analyses were performed by region, type of ACS and patient characteristics.ResultsAfter the first confirmed COVID‑19 case and during lockdown, numbers declined by up to 41% (95% confidence interval (CI): 36–47%) compared to 2019. This reduction was more pronounced for non-ST-segment elevation myocardial infarction (NSTEMI) (48%; 95% CI: 39–55%) and unstable angina (UA; 50%; 95% CI: 40–59%) than for STEMI (34%; 95% CI: 23–43%). There was no association between ACS and COVID‑19 incidence rate per region. After the steep decline, a public campaign encouraged patients not to postpone hospital visits. Numbers then increased, without a rebound effect. Trends were similar irrespective of sex, age or socio-economic status. During the outbreak, compared to coronary artery bypass graft procedures, relatively more (acute) percutaneous coronary interventions for NSTEMI and UA were performed.ConclusionNew ACS presentations decreased by up to 41%. Lockdown measures and public campaigns, rather than COVID‑19 incidence, were associated with significant changes in new ACS presentations. Even though causality cannot be established, this emphasises the role of the public media and healthcare organisations in informing patients to prevent underdiagnoses of ACS and associated health damage.Supplementary InformationThe online version of this article (10.1007/s12471-021-01603-5) contains supplementary material, which is available to authorized users.     

Survival and prognostic factors for patients with melanoma brain metastases in the era of modern systemic therapy

Historically, the prognosis of patients with melanoma brain metastases is poor, with median overall survival (OS) of 4‐6 months. Little is known of OS in the era of modern systemic therapies and local therapy with stereotactic radiosurgery (SRS) or surgery. Patients diagnosed with melanoma brain metastases at Melanoma Institute Australia from January 2011 to December 2014 were included. OS and prognostic factors were analysed using Cox regression and Kaplan‐Meier survival analyses.355 patients were included. The median OS was 7.1 months (95% confidence interval [CI] 6.0‐8.1). Median OS differed by treatment modality: systemic therapy and SRS and/or surgery 14.9 months (95% CI 10.7‐19.0), SRS and/or surgery with or without whole brain radiotherapy (WBRT) 6.4 months (95% CI 5.4‐7.5), systemic therapy 5.4 months (95% CI 3.1‐7.7), systemic therapy and WBRT 5.2 months (95% CI 4.1‐6.4), WBRT 4.4 months (95% CI 2.4‐6.3), and best supportive care 1.8 months (95% CI 1.2‐2.3). OS for patients with melanoma brain metastases appears improved in the modern era, particularly for patients who are candidates for systemic therapy with SRS and/or surgery.